Coagulation inhibition for sepsis

SummaryA well established technique to improve blood compatibility of artificial materials for use in the circulation is to coat the surface with heparin. The present report describes the antithrombin mediated inhibition of thrombin and factor Xa by surfaces modified with end point immobilized heparin. The reaction was followed by conventional chromogenic substrate based enzyme assays as well as by immunological measurement of the enzyme inhibitor (thrombin-antithrombin) complex formation. Both enzymes were rapidly inactivated by heparin surfaces after selective presaturation with antithrombin on the immobilized high affinity heparin molecules. The thrombin inhibitory capacity was enhanced when both high and low affinity heparin were preadsorbed with inhibitor. The main part of the thrombin-antithrombin complex formed remained bound to the surface, however, without functionally blocking the activity of the high affinity sequence of the immobilized heparin.Aliquots of recalcified plasma were slowly rotated in loops of heparinized tubing to investigate whether the main thromboresistant function of the surface was exerted at the level of thrombin or by inactivation of preceding enzymes. After 1 h no visible clotting occurred and only trace amounts of thrombin (0.07 IU/ml), measured as thrombin-antithrombin complexes, had been formed. In non-heparinized loops and in the test tube plasma clotted after 20 min. The thrombin generation when clotting occurred was in the order of 10 IU/ml. It is concluded that the immobilized heparin mediates inhibition of the coagulation cascade prior to prothrombin activation.

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Reactivity of Factor VIII Inhibitors in a Micro ELISA for Factor VIII : CAg and in Solid Phase Immunoisolation of VIII : CAg

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661312 ◽

1985 ◽

Vol 53 (03) ◽

pp. 346-350 ◽

Cited By ~ 8

Author(s):

O Nordfang ◽

M Ezban ◽

B Dinesen

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Solid Phase ◽

Affinity Purification ◽

Sandwich Elisa ◽

Inhibitory Capacity ◽

Sds Page ◽

As Solid Phase ◽

Peroxidase Conjugate ◽

Coagulation Inhibition

SummaryFactor VIII coagulant antigen (VIII : CAg) was measured in a sandwich-ELISA. Microplates were used as solid phase and peroxidase conjugated F(ab’)2 fragments of IgG isolated from inhibitor plasma was used as label without affinity purification. The capacity of the assay was high and the sensitivity for VIII : CAg was 0.002 U/ml. Using this assay it was possible to measure coagulation inactive VIII : CAg in samples from purification studies. Below 0.05 VIII : CAg U/ml these samples responded in parallel with standard plasma.Seven of 7 inhibitor antibodies tested were able to inhibit binding of peroxidase-conjugate in the VIII : CAg assay, and the inhibitory capacity correlated with coagulation inhibition as measured by the Bethesda method. Using the highest titered antibodies bound to a solid phase, VIII : CAg was isolated and identified in SDS-PAGE as a doublet with a molecular weight of 77-80 kD.

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HUMAN AND PORCINE FVIII INHIBITOR RESPONSE FOLLOWING INFUSION OF HUMAN FVIII CONCENTRATE - MEASUREMENT USING AN AGAROSE GEL SYSTEM

10.1055/s-0038-1644056 ◽

1987 ◽

Author(s):

D J Perry ◽

F G H Hill

Keyword(s):

High Responder ◽

Agarose Gel ◽

Haemophilia A ◽

Single Treatment ◽

Fviii Inhibitor ◽

Antibody Titres ◽

Appropriate Therapy ◽

Gel Technique ◽

Mild Haemophilia ◽

Coagulation Inhibition

Conventional methods for determining inhibitor levels in haemophiliacs are time consuming and labour intensive. The agarose gel technique of Jorquera et al. (1) has been modified and standardized to measure human and porcine inhibitors to VIII:C.26 samples from 12 haemophiliacs with inhibitors were analysed and in all cases antibody to human and porcine VIII:C was detected.Six haemophiliac patients with 'high responder-type' inhibitors were studied using stored plasma and the rise in antibody titres to both human and porcine VIII:C was determined sequentially during treatment with human FVIII concentrate.One patient (J. C.) had received a single treatment with porcine concentrate (Hyate:C) with no rise in porcine VIII:C.In 5 of 6 patients both porcine and human inhibitor titres rose but the porcine levels were less than the human. However, in the remaining high responder patient (I. I.) with previously mild haemophilia and no exposure to porcine FVIII, the rise in porcine antibody titre greatly exceeded that of human. This patient has continued to be treated with human FVIII concentrate during which time the human inhibitor titres have fallen more than the porcine.This method is simple to perform and has the advantage that both human and porcine inhibitor titres can be easily and quickly assessed so that the most appropriate therapy can be given.1. Jorquera JI, Carmona E, Aznar JA, Peiro A and Sanchez-Cuenca JM. (1985) A Standardized Method for Measuring Anti-FVIII:C Inhibitors in Haemophilia A by Coagulation Inhibition in Agarose Gel. Thromb. Haemostas. 54(2): 377-380.

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